Diabetic retinopathy (DR) is a leading cause of blindness in the U.S. The proliferative type (PDR) involves severe inflammatory and angiogenic responses resulting in retinal degeneration. Vascular damage is followed by a burst of angiogenesis that rapidly creates an even more complex capillary network of leaky and fragile vessels. We have detected a similar process in Matrigel cultures of retinal endothelial cells (REC). It is well known that endothelial cells plated on this matrix 1) stop proliferating, 2) form capillary-like tubes for 24-48 hr, 3) do not invade the matrix, 4) collapse into clumps, and 5) die. Because this was thought to be the endpoint of the assay, no experiments extended beyond this point. However, we report the discovery of spontaneous cell survival following tube collapse. These surviving cells 1) proliferate, 2) migrate, 3) form spherical colonies that remain alive for weeks, 4) invade the matrix, and 5) can reassemble into larger tubes. We have named this novel phenomenon "secondary sprouting". Since the angiogenic phenotype of surviving colonies is markedly different from the initial cells, we hypothesize that these behaviors are caused by changes in the cells themselves and/or in the surrounding matrix. If diabetes induced cell injury promotes the sprouting phenotype, simulating such injuries in vitro should increase secondary sprouting. Consequently, cultures from diabetic and diabetic retinopathy REC should exhibit a higher sprouting capacity than those from normal patients. Specific aim #l: Determine if the "pre- and post-collapse" extracellular matrices are different and whether these alterations induce the sprouting phenotype of "post-collapse" colonies. Specific aim #2: To determine if the "pre- and post-collapse" cells are different and whether these differences are temporary or permanent. If changes are temporary, we will examine whether growth factor and metalloproteinase production results in the sprouting phenotype; if the changes are permanent, we will determine if they are due to terminal differentiation of the whole cell population or to selection of a specific subpopulation. Specific aim #3: Investigate whether simulation of diabetic cellular injuries enhances the secondary sprouting ability of REC. Specific aim #4: Determine if REC derived from NL, DM and DR patients have differential abilities to form secondary sprouts. These experiments could explain how cellular injury induces blood vessel regression as well as the subsequent expansion of the capillary bed. The discovery and modulation of molecules involved in secondary sprouting could provide novel and effective therapeutics for PDR and other conditions associated with neovascularization.